We propose to study two major mechanisms that regulate the structure and function of the plasma membrane: ER quality control and lipid rafts. We have used mutants of PMA1 encoding the yeast plasma membrane H+ATPase as tools to understand normal membrane biogenesis as well as the molecular mechanisms of regulation at the ER and at the plasma membrane. The dominant negative Pma1-D378N mutant is recognized and targeted for destruction by the ER-associated degradation (ERAD) pathway, whereas the temperature-sensitive Pma1-10 mutant fails to associate with lipid rafts and is removed from the plasma membrane for vacuolar degradation. Each pma1 mutant has been used as the basis for genetic selection leading to identification of Eps1, a novel component of the ERAD pathway, and Yvh1, a dual-specificity phosphatase, which may play a role in quality control at the cell surface. This proposal integrates biochemical, cell biological and genetic approaches to study the molecular mechanisms of these novel proteins. Specific Aim I addresses a proposed role for Eps1, a member of the protein disulfide isomerase family, in substrate recognition during ERAD. In addition, the ERAD substrate, Pma1-D378N, will be analyzed in detail because it represents a major resource for studying ERAD. Specific Aim II uses the pma1-10 mutant and its suppressor yvh1 to study the relationship between protein phosphorylation, ubiquitination, association with lipid rafts, and protein stability. Our studies have important implications for understanding human diseases caused by misfolding and ERAD of important molecules, including cystic fibrosis. Our results on lipid rafts will have significance for understanding the immune response (during which signaling occurs in rafts), AIzheimer's disease (in which protein processing is raft-based), and lipid storage diseases (in which raft lipids are abnormally accumulated in lysosomes).